The Seeds of Cancer

Penn SBDRC-supported study identifies mutations and biomarkers associated with carcinoma in situ

  • Mutations and biomarkers were examined in patient biopsies taken in the early stages of squamous cell carcinoma.
  • Laser capture microdissection was coupled with exome-seq and RNA-seq of patient biopsies.
  • The data provide new insights regarding the mutations that drive the formation of squamous cell carcinoma in situ and the expression of genes associated with the formation of these lesions.
  • The study was a collaboration among the SBDRC Cores, led by Dr. John Seykora, Director of CPAT (Cutaneous Phenotyping and Transcriptomics) Core. The first author, Dr. Qi Zheng, is a Technical Director of the DSI (Data Sciences and Informatics) Core.

PHILADELPHIA – For decades, medical experts have known that UV irradiation causes mutations in skin cells that lead to cancers including squamous cell carcinoma (SCC). SCCs are known to contain more mutations than almost all other forms of cancer; however, many of the mutations identified in SCCs are “passenger” mutations, though present, are not thought to contribute to SCC formation. This situation makes it difficult to extrapolate  which mutations present in SCCs are important for driving the early stages of UV-induced skin cancer called squamous cell carcinoma in situ (SCCIS). To identify the mutations associated with SCCIS, a team of Penn Medicine researchers led by John T. Seykora, MD, PhD, Professor of Dermatology in the Perelman School of Medicine at the University of Pennsylvania, utilized an innovative approach to analyze SCCIS samples by employing laser-capture microdissection technology which combines the cutting precision of lasers coupled with the high visual resolution of a microscope. With this equipment, the investigators were able to precisely identify and isolate SCCIS cells and cut them out of the tissue samples to collect them. In parallel, the investigators also dissected skin cells from adjacent unremarkable epidermis and collected them to serve as a control sample. Their findings were recently published in the Journal of Investigative Dermatology.

Next, the investigators isolated the DNA and RNA from the micro-dissected samples and prepared the DNA for whole-exome sequencing and the RNA for RNA sequencing. In this way, they were able to sequence all the protein-coding elements in the SCCIS and skin cells to identify mutations present in the SCCIS cells that were not present in the unremarkable skin cells. Using this approach, they identified specific mutations associated with the SCCIS which provided novel insights into how these lesions develop. This sequencing data revealed that loss-of-function mutations in NOTCH 1-3 were found in the UV-exposed skin and a subset of such mutations demonstrated evidence of positive selection in the SCCIS. The investigators also showed that oncogenic mutations in the TP53 gene, a master regulator of DNA integrity, were present in the SCCIS but not in the adjacent skin. This observation suggests that skin cells may acquire a NOTCH mutation first followed by a TP53 mutation during formation of an SCCIS. Another major finding was that no oncogenic mutations in RAS genes were found in SCCIS, though they have been reported in approximately 15% of SCCs. This observation indicates that oncogenic RAS mutations, which is one of the most commonly mutated genes all cancers, are not found in the early but latter stages of SCC development.

The investigators also determined the gene expression profile for SCCIS cells and corresponding unremarkable skin cells. The RNA sequencing data showed that there were 1166 differentially expressed genes between SCCIS and epidermis and that the largest class of differentially expressed genes were those the regulate the immune system. Another gene found to be upregulated in SCCIS was NEURL1 (Neuralized 1) which is a ubiquitin-ligase that downregulates proteins that are known to activate the Notch signaling pathway. The investigators confirmed that the protein level for Neurl1 was increased in SCCIS compared to adjacent skin using protein immunohistochemistry. These data implicate another pathway that may inhibit Notch signaling by downregulating proteins known to activate Notch. Together, the data from this study provide new insights regarding the mutations that drive the formation of SCCIS and the expression of genes associated with the formation of these lesions; these data could provide new therapeutic targets for treating SCCIS.

The research team included first author Dr. Qi Zheng, a Senior Bioinformatician and Dr. Brian Capell, Assistant Professor of Dermatology. Additional Penn authors of the study include Vishwa Parekh, Conor O’Day, Cem Attilasoy, Hasan Bashir, Christopher Yeh, Eun-Hee Shim, Stephen Prouty, Tzvete Dentchev, Vivian Lee, Lily Wushanley, Yerin Kweon, Yoko Suzuki-Horiuchi, Warren Pear and Elizabeth Grice.

The work was supported by the National Institutes of Health (R01 CA165836, P30-CA016520, P30 AR06958901, R01 CA163566, RO1 ES013508 and P30CA016520, K08 EY025742).


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